1. Field of the Invention
The invention relates to peptides useful as vaccines for the prophylaxis and/or treatment of Human Immunodeficiency Virus infection in humans, to compositions incorporating such peptides and to methods for the administration of such vaccines.
2. Related Art
This application makes reference to various literature publications, which are herein incorporated in their entirety by reference.
Live virus vaccines and killed whole or subunit virus vaccines for AIDS have potential safety risks. In contrast, synthetic peptides are inherently safe. Furthermore, molecules corresponding to whole viral proteins but made by recombinant DNA technology contain, in addition to protective epitopes, structures which potentially will elicit suppression of the immune response, or which will elicit antibodies that, rather than being protective, may enhance viral uptake and thus be deleterious. A vaccine which contains only selected peptides that elicit the appropriate type of immunity and do not have other deleterious effects should be more effective for a difficult virus such as HIV.
Our previous work showed that the major CTL antigenic determinant of HIV-1 envelope protein gp160 consisted of residues 315-329 in the numbering sequence of Ratner et al. (27), However, we have now found that this peptide does not bind intact to the class I MHC molecule that must present it to CTL, but rather it must first be proteolytically cleaved, by proteases such as those present in serum.
T-cell stimulation by the HIV-1 gp160 -derived p18 peptide presently by H-2D.sup.d class I major histocompatibility complex (MHC) molecules in a cell-free system was found to require proteolytic cleavage. This extracellular processing was mediated by peptidases present in fetal calf serum (FCS). In vitro processing of p18 resulted in a distinct reverse phase HPLC profile, from which a biologically active product was isolated and sequenced. This peptide processing can be specifically blocked by the angiotensin converting enzyme (ACE) inhibitor captopril and can occur by exposing p18 to purified ACE. The ablity of naturally occurring extracellular proteases to convert inactive peptides to T-cell antigens has important implications for understanding cytotoxic T-lymphocyte (CTL) responses in vivo and for rational peptide vaccine design.
Although naturally processed peptides associate with newly formed MHC class I molecules intracellularly (1), extracellular loading of surface class I molecules by synthetic peptides (2) is commonly used to analyze MHC class I peptide interactions. Recent data have provided substantial evidence that peptides bound to class I are approximately nine amino-acids in length (3-9), but larger peptides are capable of sensitizing targets for class I MHC-restricted lysis. In some cases the activity of these longer peptides can be traced to the prsence of contaminating shorter products which are extremely biologically potent (9).
The HIV-1 (IIIB) gp160 envelope glycoprotein-derived peptide, p18, is 15 amino acids in length (residues 315-329). It is the immunodominant CTL determinant of gp160 in H-2D.sup.d mice (10,11) and can sensitize syngeneic cells for lysis by CTL from HIV-1-infected humans (12).
If a person suspected of being exposed to AIDS is tested for antibodies to HIV and is determined to be seronegative, it is still possible that that person is carrying the virus but has not made antibodies, because a certain percentage of exposed individuals do not develop antibodies for a significant period of time after exposure, and some may never develop antibodies. Nevertheless, that person may have developed a cell-mediated immune response in the form of CTL specific for the envelope of the virus. Because that person's CTL are specific not only for the virus but also for that individual's major histocompatibility complex (MHC) antigens, it is not possible to test for these on transfected tumor targets as was done in the case of the mice, unless one is lucky enough to have transfected tumor that shares MHC (HLA) molecules with the individual to be tested. Because there are so many human HLA types, it is not feasible to have transfected cells of every type available. Although it is possible to produce a transformed tumor line from the individual and transfect it with HIV genes, this is a very difficult, time consuming, laborious process and would not be feasible to do for large numbers of people. Infecting the individual's cells with HIV requires the appropriate cell type that can be infected, and would be hazardous for laboratory workers to handle the concentrated virus. Use of the vaccinia recombinant described above may give many false positive results since most individuals in the U.S. born before 1972 were immunized with vaccinia as a smallpox vaccination, and so would have vaccinia-specific CTL. Purified proteins are generally not taken up by cells in such a way as to make them targets for CTL. However, small peptides such as P18 IIIB (earlier called Env-K.sub.1) are capable of sensitizing targets for CTL, as we have shown herein above. Therefore, it would be relatively simple to use P18 IIIB as a diagnostic reagent to test for the presence of HIV-specific CTL in the peripheral blood of an individual. This can be achieved by standard procedures; first, produce PHA or ConA blasts of the peripheral blood lymphocytes of the individual to be tested, label these with .sup.51 Cr as indicated herein above, incubate these with the peptide P18 IIIB under standard culture conditions similar to those given herein above, but modified for human CTL assays, and add fresh peripheral blood lymphocytes from the same individual. After about 6 hours, one measures the amount of .sup.51 Cr released into the culture medium, and compares this with controls treated identically but without any peptide, or with a control peptide, without any fresh lymphocytes, and with the maximum .sup.51 Cr release produced by detergent lysis of the target cells. If there is specific release, the individual can be judged to be carrying the HIV virus, even though no antibodies could be detected.
Previous studies of the ability of this peptide to form stimulatory complexes with purified H-2D.sup.d molecules in vitro, indicated that two activities of FCS were required for recognition of p18 by a specific T-cell hybridoma. One activity was that of .beta.2-microglobulin (.beta.2-m) (13, 14-17) and the other activity could be performed by ovalbumin. Most batches of bovine serum albumin (BSA) were unable to replace this .beta.2-m independent effect of FCS.
We have tested 9, 10 and 11 residue peptides, derived from p18, overlapping or contained within the p18-I-10 peptide, including specifically both possible 9 residue peptides contained within p18-I-10, and all of these have been found to be less active than p18-I-10. This finding concerning the importance of length in the activity of peptides presented by MHC class I molecules and the identification of a truncation of p18, p18-1-10 (residues 318-327), with 10 to 10.sup.2 -fold greater potency of T-cell stimulation prompted us to consider the possibility that ovalbumin and FCS were processing p18 to an active, shorter peptide.
Cytotoxic T lymphocytes (CTL) and T helper cells recognize processed antigenic peptides in association with the products of the major histocompatibility complex (MHC) (26-30). Generally, CD8+ CTL are restricted by MHC class I molecules, such as H-2K, -D, -L in mice and HLA-A, -B, -C in humans, presented on the surface of antigen-presenting cells (APC), while CD4+T helper cells (Th) are restricted by MHC class II molecules, such as I-A or I-E in mice and HLA-DR, -DQ or -DP in humans. T cells are able to recognize a wide variety of antigens in the context of relatively few MHC molecules by means of specific T cell receptors (TCR) (31-34). There is no known difference in overall TCR repertoire between CD4+ and CD8+ T cells.
Although it has generally been assumed that there is no reason to expect the same peptides to be presented by both class I and class II MHC molecules, there are a few cases reported in which peptides presented by class I molecules were found to be presented by or to bind to class II molecules also (35,36). Moreover, we have recently found that the immunodominant antigenic determinant of HIV-I envelope protein gp160 recognized by BALB/c murine as well as human CD8+ CTL with class I MHC molecules (peptide P18IIIB, residues 315-329, RIQRGPGRAFVTIGK, SEQ. ID. NO. 1) (37,38), is also presented by class II MHC molecules of both mice (39) and humans (40) to CD4+ helper T cells. Conversely, we found that three other peptides of HIV-1 gp160 that were originally identified as stimulating CD4+ helper T cells of mice (41,42) and humans (40,43) also were presented by human class I molecules to human CD8+ CTL (38). Thus, we asked whether these latter peptides also were presented by murine class I molecules to CD8+ CTL, and if so, what range of class I molecules could present them.
These findings also led us to raise a related but distinct question. A few cases have been described of antigenic determinants that happen to be broadly or permissively presented by multiple class II MHC molecules, especially in the case of murine I-E or human DR, in which polymorphism is limited to the beta chain, but the alpha chain is conserved (44,45). However, no similar cases have been studied for presentation by class I MHC molecules, and no analysis of 10 different class I MHC haplotypes as here has previously been reported. Because both domains of the MHC peptide-binding site are polymorphic in class I molecules, exploring permissiveness in class I presentation would be of interest in comparison with class II. Also such widely presented antigenic determinants would clearly be useful for development of synthetic vaccines aimed at a broad outbred population of diverse MHC types. This is especially relevant for HIV-1, because whole virus and even whole envelope protein can elicit deleterious immune responses that can enhance infection or contribute to the development of immune deficiency (reviewed in (46)).
Therefore, for both theoretical and potential practical interest, we explored the breadth of presentation by class I MHC molecules from ten distinct murine MHC haplotypes of both the original CTL determinant peptide P18, and two of the original helper T-cell determinant peptides Tl (428-443, KQIINMWQEVGKAMYA, SEQ. ID. NO. 15), and HP53 (HP53, 834-848, also known as TH4.1, DRVIEVVQGAYRAIR, SEQ. ID. NO. 16). P18 and HP53 were presented by at least 4 different class I MHC molecules in mice immunized with recombinant vaccinia virus transfected with HIV-1 gp160, and Tl was recognized by CD8+ CTL in mice of three MHC haplotypes. Indeed, even the same segments of the peptides are recognized by the several haplotypes. Thus, permissiveness of presentation by class I molecules appears to be at least as great as that reported for presentation by class II molecules, and the extent of overlap between the repertoire of sites presented by class I and the repertoire of sites presented by class II may be much greater than suspected. Also, from a practical point of view, these peptides that are broadly presented by multiple class I as well as class II MHC molecules may be versatile components of a vaccine.